DNA amplification technology has developed rapidly in recent years as researchers have discovered its value for detection of nucleic acids that are present in small quantities in test samples. The use of probes is based upon the concept of complementarity. DNA has two strands bound together by hydrogen bonds between complementary nucleotides.
The DNA complex is normally stable, but the strands can be separated (or denatured) by conditions that disrupt the hydrogen bonding. The released single strands will reassociate only with another strand having a complementary sequence of nucleotides. This hybridization process can occur with both strands being in solution or with one of the strands being attached to a solid substrate.
A targeted nucleic acid sequence in an organism or cell may be only a very small portion of the entire DNA molecule so that it is very difficult to detect its presence using most labeled DNA probes. Much research has been carried out to find ways to detect only a few molecules of a targeted nucleic acid.
Such techniques, which generally involve the amplification and detection (and subsequent measurement) of minute amounts of target nucleic acids (either DNA or RNA) in a test sample, include inter alia the polymerase chain reaction (PCR) (Saiki, et al., Science 230:1350, 1985; Saiki et al., Science 239:487, 1988; PCR Technology, Henry A. Erlich, ed., Stockton Press, 1989; Patterson et al., Science 260:976, 1993), ligase chain reaction (LCR) (Barany, Proc. Natl. Acad. Sci. USA 88:189, 1991), strand displacement amplification (SDA) (Walker et al., Nucl. Acids Res. 20:1691, 1992), Q.beta. replicase amplification (Q.beta.RA) (Wu et al., Proc. Natl. Acad. Sci. USA 89:11769, 1992; Lomeli et al. Clin. Chem. 35:1826, 1989) and self-sustained replication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990).
The standard PCR method relies on two primers, one forward and one reverse. The combination of the two primers allows for the exponential amplification of particular DNA targets and a means for continuously generating DNA fragments of specific sizes. A major problem with PCR is having to continually denature DNA at 95 C, a situation that would cause the DNA polymerase to rapidly lose activity. In U.S. Pat. No. 5,849,497 (Steinman et.al.) a process is described for inhibiting the amplification of a DNA template by subjecting a sample of biological material containing nucleic acid to PCR using a DNA polymerase deficient in 5′exonuclease activity (i.e., Stoffel Taq polymerase).
The present invention describes a novel method of blocking DNA amplification, to allow for the amplification of DNA. The unique features of the present invention include: 1) the use of one primer instead of using two primers, 2) the use of a blocker DNA molecule to generate primer extension products of specified length, i.e., on the basis of the hybridization site of the non extendable oligonucleotide blocker on the DNA template relative to the up stream primer binding site, and 3) the use of a chimeric primer molecule comprising a sequence of deoxyribonucleotides with a 3′ ribonucleotide terminus end to allow, in the presence of RNAse H, a continuous source of primer extension products of specific sizes.
Unlike other DNA amplifications methods which require multiple denaturation steps at 95 degrees Celsius, the method of the present invention requires denaturation of the DNA template at 95 degrees Celsius only once at the start of the procedure.
The utilization of chimeric primers to amplify nucleic acids or to obtain transcripts of a nucleic acid has been the subject of several patents and publications. Such a method is disclosed by the International Patent Application WO 00/56877 (Mukai et. al.) that describes using an endonuclease (RNAse H), a DNA polymerase enzyme and a chimeric oligonucleotide primer with a ribonucleotide on the 3′ terminal end or on the 3′ termination side to amplify DNA What distinguishes the Mukai et. al. method from the present invention is that in the Mukai procedure the method depends strictly on a DNA polymerase with a strand displacement activity. U.S. Pat. No. 5,744,308 (Guillou-Bonnici et. al.) describes an invention requiring a chimera oligonucleotide that can be used in a process for obtaining transcripts and/or amplification of a target sequence of a nucleic acid, having, at its 3′ end, a downstream sequence. The oligonucleotide comprises successively, from 5′ to 3′, 1) a first oligonucleotide segment, of the DNA type, comprising a sense sequence of a promoter of an RNA polymerase, 2) a second oligonucleotide segment, of the DNA type, capable of hybridizing with the downstream sequence, and 3) a third oligonucleotide segment, of the RNA type, capable of hybridizing with a part of the target sequence contiguous to the downstream sequence, the third segment being blocked at 3′. A process using the chimera oligonucleotide and an enzyme system containing DNA polymerase activity, RNA polymerase activity, and a third activity, for example, an RNAse H activity provides transcription products of the target. By adding a second chimera oligonucleotide capable of hybridizing with the complement of the target, cyclic amplification of the target and its complement are obtained. The method of Guillou-Bonnici is different from the present invention by several aspects. First, the present invention has no need for an oligonucleotide segment, of the DNA type, comprising a sense sequence of a promoter of an RNA polymerase. Secondly, the method of Guillou-Bonnici et. al. was specifically tailored to amplify RNA and not DNA products.
U.S. Pat. Nos. 4,876,187 and 5,011,769 (Duck et. al. and Bekkaoui et. al.), disclose a cycling probe method that employs probes comprising RNA, preferably DNA: RNA: DNA chimeras.
Thus, there exists a need for an effective method to amplify DNA without the continuous thermocycling to high temperatures. The present invention satisfies this need and provides related advantages as well. The possible uses of the present invention, described above, are only examples and are not meant to limit the scope of the present invention in any way.